Nucleic acid purification

ABSTRACT

Methods and composition for nucleic acid isolation are provided. In one embodiment, a method is provided for nucleic acid purification from biological samples, such as whole blood samples, extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for of small RNAs (e.g., siRNAs or miRNAs) purification and are amenable to automation.

The present application is a continuation of U.S. patent applicationSer. No. 13/943,986, filed Jul. 17, 2013, which claims the prioritybenefit of U.S. provisional application No. 61/673,106, filed Jul. 18,2012, which is incorporated herein by reference in its entirety.

This application is also related to U.S. patent application Ser. No.13/349,020, filed Jan. 12, 2012, now U.S. Pat. No. 9,051,563, and U.S.Provisional Patent Application No. 61/432,749, filed Jan. 14, 2011, eachof which is incorporated herein by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention generally relates to biochemistry and molecularbiology. More specifically, the invention relates to methods andcompositions for purification of nucleic acid molecules.

2. Description of the Related Art

A variety of protocols have been developed for purification of nucleicacids. One crucial step in many purification protocols involves theseparation of nucleic acids from protein and lipid molecules thatconstitute cells and tissue matrices. Once denatured, proteins aretypically highly hydrophobic, while nucleic acids remain hydrophilic.Accordingly, organic solvents, such phenol, have been widely used tosolubilize proteins and lipids associated with nucleic acids into anorganic phase. However, phenol-based reagents must later be carefullyremoved from any nucleic acid preparation because phenol is toxic andinterferes with downstream processes (such as sequencing orhybridization) that may be used to analyze nucleic acid.

SUMMARY

In a first embodiment, the invention provides a method for binding anucleic acid molecule to a substrate in the presence of phenol. Forexample, the method can comprise (a) obtaining sample comprising anucleic acid molecule and phenol and (b) contacting the sample to asilica substrate in the presence of a binding agent comprising achaotropic salt, an alcohol or a combination thereof, thereby bindingthe nucleic acid molecule to the silica substrate. In certain aspects, anucleic acid containing sample may comprise a substantial amount ofphenol, such as about or greater than about 10%, 20%, 30%, 40% or 50%phenol by volume.

In a further embodiment, the invention provides a method for nucleicacid purification comprising (a) contacting a nucleic acid containingsample with a denaturing solvent comprising phenol; (b) adding a bindingagent to the sample, wherein the binding agent comprises a chaotropicsalt, an alcohol or a combination thereof; and (c) contacting the samplewith a silica substrate thereby binding the nucleic acid to the silicasubstrate. A nucleic acid purification method according to the inventionmay comprise performing steps (a)-(c) in any order. Alternatively oradditionally, two or more of steps (a)-(c) may be performed essentiallycontemporaneously. For example, a binding agent and silica substrate canbe added to a sample contemporaneously (e.g., as part of the samereagent) or a binding agent may be added to a sample prior to contactingthe sample with a denaturing solvent comprising phenol. In still furtheraspects, steps (a) and (b) may be performed contemporaneously, forexample, by contacting the sample with the denaturing solvent comprisingphenol and the components of a binding agent.

In certain embodiments, the nucleic acid in a sample comprising phenolare substantially unprecipitated prior to binding of the nucleic acid tothe silica substrate. For example, in certain aspects, prior to bindingof the nucleic acid to the substrate, at least about 90% of the nucleicacid remains in solution in the sample. In certain instances, a samplecomprising phenol may comprise an aqueous and an organic phase. Thus, inone aspect of the invention, the nucleic acid is bound to the silicasubstrate with out substantially separating the organic and aqueousphases in the sample (e.g., without removing a substantial portion ofone phase or the other from the sample prior to the binding). Thus, insome aspects, a method of the invention may be defined as a method fordirectly binding a nucleic acid molecule to silica substrate from anaqueous/organic suspension. In further aspects, a method according tothe invention does not comprise a centrifugation step (such as a step toform a nucleic acid pellet by centrifugation) prior to the binding ofthe nucleic acid to the silica substrate. Thus, in certain aspects, amethod according to the invention may be defined as a method for bindingat least about 50%, 60%, 70%, 80%, 90%, 95% or more of the nucleic acid(e.g., RNA and/or DNA) from a sample to a silica substrate.

In certain embodiments, methods of the invention involve denaturingsolvents comprising phenol (or carbolic acid). For example, in certainaspects, a denaturing solvent comprises greater than about 30% phenol byvolume, such as at least about 40%-60% phenol. In some aspects, suchdenaturing solvents are acidic, having a pH of less than about 6.0, 5.5,5.0, or 4.5 (e.g., a pH of about 4.0). In certain aspects a denaturingsolvent has a pH of between about 6.0, 5.5, 5.0, 4.5, 4.0 and about 3.5,3.0, 2.5 or 2.0. Thus, in some cases, denaturing solvents comprisingphenol are equilibrated with an aqueous buffer to a desired acidic pH,such as a pH of less than 5.0 (e.g., between about 5.0 and about 3.0).Denaturing solvents may further comprise additional ingredientsincluding, but not limited to, chloroform, isoamyl alcohol, guanidiniumthiocyanate, guanidinium chloride, BCP (1-bromo-3-chloropropane) BAN(4-bromoanisole), an antioxidant (e.g., 2-mercaptoethanol), a chelatingagent (e.g., EDTA or EGTA) or isoamyl alcohol. For example, an agent maycomprise phenol, guanidinium thiocyanate and other components at a ratioof about 5:3:2. For example, a denaturing solvent can be selected fromone of those provided in U.S. Pat. No. 4,843,155, incorporated herein byreference in its entirety. Commercially available denaturing solventsthat may be used according the invention include, but are not limitedto, TRI Reagent®, RNAzol® (Molecular Research Center), TRIzol®(available from Invitrogen) and Qiazol® (available from Qiagen).

Some aspects of the invention concerns the addition of at least a firstbinding agent to a sample to facilitate nucleic acid binding to a silicasubstrate. For example, the binding agent may comprise an alcohol suchas a lower alcohol, e.g., methanol, ethanol, isopropanol, butanol,1-methyl-3-butanol or a combination thereof. In some embodiments thebinding agent can comprise Dioxane which is used similarly to orsubstituting for alcohols including lower alcohols (e.g., 1,4-Dioxane orrare 1,2 or 1,3 isomers of Dioxane). In some embodiments, the bindingagent is a substantially aqueous solution comprising a chaotropic salt.For example, the binding agent may be an aqueous solution comprising abuffering agent, an alcohol, a chaotropic salt, a chelating agent and/ora detergent. A binding agent can be added to a sample comprising nucleicacid before, after or contemporaneously with the contacting of thesample to a silica substrate. Thus, in some aspects, a binding agent mayfurther comprise a silica substrate, such as a slurry of silica beads.

In certain embodiments, the binding agent may be a substantially aqueoussolution comprising one or more chaotropic salts and, optionally, analcohol. For example, a binding agent may comprise a chaotropic salt(e.g., guanidinium thiocyanate, guanidinium hydrochloride sodium iodide,sodium perchlorate, urea or thiourea) or a mixture of chaotropic saltsand an acetate salt (e.g., sodium acetate) and may, or may not, comprisean alcohol. In certain aspects, a binding agent comprises aconcentration of an acetate salt such that, after addition of thebinding agent, the sample comprises a concentration at least about 100mM (e.g., about 0.1 M to 1.0 M) relative to the acetate salt.Alternatively or additionally, a substantially aqueous binding agentcomprises an alcohol such that the total alcohol content of the sampleafter the binding agent is added is about 2.5% to about 40% alcohol. Insome aspects, a substantially aqueous binding agent comprises at leastor at most about 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5,20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5,26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5,32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5,38.0, 38.5, 39.0, 39.5, 40.0 percent alcohol or any range derivabletherein. For example, the binding agent can comprise about 20%-40%,about 20%-30% alcohol, about 2.5%-25%, about 2.5%-20% alcohol or about5%-25% alcohol. Such aqueous binding agents may be used, for example, tobind DNA molecules to a silica substrate. In still other embodiments thebinding agents can comprise Dioxane such that the total Dioxane contentof the sample after the binding agent is added is about 2.5% to about30% Dioxane (e.g., about 2.5%-25%, about 2.5%-20% Dioxane or about5%-25% Dioxane). In yet further aspects, a substantially aqueous bindingagent comprises about 20%-30% or about 20%-40% Dioxane or about 40%-100%Dioxane. Such aqueous binding agents may be used, for example, to bindDNA molecules to a silica substrate.

In further embodiments, the binding agent may be a substantially alcoholbinding agent such as an agent that increases the total alcohol contentof the sample to greater than about 40% after addition (e.g., an agentthat increases the total alcohol content of the sample to greater thanabout 45%, 50%, 55% or 60%). For instance, a substantially alcoholbinding agent may be a solution that is 80%, 90%, 95%, 98% oressentially 100% alcohol, such as a lower alcohol (e.g., ethanol). Suchalcohol-based binding agents may be used, for example, to bind RNA orRNA and DNA molecules to a silica substrate. In certain aspects, thebinding agent may further comprise a phase separating agent.

In a further embodiment, a method of the invention comprises one or moresteps selected from the group consisting of: adding a phase separatingagent to a sample; removing the sample suspension from the silicasubstrate after binding of the nucleic acid; contacting a silicasubstrate bound nucleic acid with a wash agent (e.g., washing the silicasubstrate); and eluting the nucleic acid from the silica substrate withan elution buffer. For example, a phase separating agent, such as anagent comprising chloroform, BCP (1-bromo-3-chloropropane) or BAN(4-bromoanisole) can be added. Wash agents for use according to theinvention may, for example, comprise an aqueous solution with high saltand/or alcohol content and optionally may include detergents. Examplesof elution buffers include, but are not limited to, aqueous solutionssuch as solutions comprising a buffering agent, a chelating agent and/orone or more nuclease inhibitors.

Methods according to the invention may be used for the purification ofDNA (e.g., genomic or plasmid DNA), RNA or a combination thereof. Incertain embodiments, a method of the invention is used for the isolationof RNA and the binding agent comprises a substantially alcohol bindingagent. In another embodiment, a method according to the invention isused for DNA purification and the binding agent comprises asubstantially aqueous buffer (e.g., an aqueous buffer comprising achaotropic salt, an alcohol and, optionally, a detergent).

In still a further embodiment, a method according to the invention maybe used to sequentially purify DNA and RNA. For instance, an aqueousbinding agent comprising a chaotropic salt may be added to a samplecomprising a nucleic acid and the suspension contacted with a firstsilica substrate to bind DNA from the suspension. In one example, anaqueous binding agent for DNA binding additionally comprises an alcoholand can be used to raise the alcohol content of the suspension to about5-20%. In a second example, an aqueous binding agent for DNA bindingadditionally comprises an acetate salt and can be used to raise theacetate content of the sample to about or above about 0.1 M, 0.2 M, 0.3M, 0.5 M, 0.75 M, or 1.0 M. Following removal or collection of thesilica-bound DNA, the suspension can then be mixed with a second bindingagent, such as a substantially alcohol binding agent and contacted witha second silica substrate to bind RNA from the suspension. For example,the second binding agent for RNA binding can be used to raise thealcohol content of the suspension to greater than about 50%. DNA and RNAmay then be eluted from the first and second silica substratesaccordingly. Thus, methods for preferentially purifying DNA and RNA fromthe same sample suspension are also provided by the instant invention.

In certain aspects, methods according to the invention concern a silicasubstrate. A variety of silica substrates may be used according to theinvention including, but not limited to, silica beads (e.g., magneticbeads), fibers, silica plates or a porous silica matrix. In certainaspects, a silica substrate is immobilized such that a suspension or asolution can be moved over or through the substrate. For example, asilica substrate may be comprised in a column or in an array of columns(see, e.g., U.S. Patent Publication 20100222560, incorporated herein byreference). In certain aspects, a column for use according to theinvention is adapted such that suspensions or solutions can be pushed(e.g., by applying pressure via a pipette) or pulled (e.g., by a vacuumpressure) through a column. Thus, in certain aspects, a method fornucleic acid purification according to the instant invention does notcomprise a step involving centrifugation. A variety of materials may beused to manufacture a column for use according to the methods of theinvention. In certain aspects, the column or the interior surfacesthereof are composed of a material that is substantially resistant todegradation by phenol (e.g., polypropylene). In still further aspects, acolumn for use according to embodiments is comprised in an array ofcolumns such as in an array of 96 or 384 columns (e.g., adapted for usewith a corresponding 96- or 384-well microtiter plate).

A sample for use according to the invention may be any sample thatcomprises or potentially comprises a nucleic acid. For example, thesample may comprise genomic DNA, plasmid DNA or RNA. For instance thesample may comprise RNA, such as mRNA or rRNA or small RNA molecules. Asused herein a small RNA molecule refers to a molecule less than about200 nucleotides (e.g., less than about 150, 100 or 50 nucleotides) inlength. Examples of such molecules include, without limitation, smallinterfering RNAs (siRNAs), micro RNAs (miRNAs), piwi RNAs (piRNAs), aswell as small synthetic RNAs, such as RNA oligonucleotides. In furtheraspects, a sample of the embodiments is a sample that is substantiallyfree of larger RNAs (RNAs over 200 nucleotides in length) and/or free ofDNA. In some aspects, a method of the embodiments is further defined asmethod for isolating or purifying a small RNA.

A sample can be obtained from a variety of sources such as from ananimal subject, a plant or from a cell line or tissue bank. A sample maybe a fresh sample or a frozen or desiccated sample. For example a samplefrom an animal may be a blood sample (e.g., a whole blood sample), aurine sample, a fecal sample, a tissue sample (e.g., a biopsy), a salivasample, or a hair sample.

In certain embodiments, methods according to the invention may furthercomprise one or more treatment steps prior to binding of nucleic acid toa silica substrate. In certain aspects, a sample, such as a tissuesample, can be disrupted prior to treatment with denaturing solvent. Asample can, for example, be disrupted mechanically by chopping orgrinding or enzymatically (e.g., by proteinase digestion). In someaspects, a method involves a step for shearing genomic DNA in the sampleprior to binding of nucleic acid to a silica substrate. For example, theshearing can be mechanical shearing (e.g., repeated pipetting orsonication) or limited enzymatic digestion.

In still a further embodiment, a method according to the invention maybe defined as a method for purifying nucleic acid from a pluralitysamples. For example, nucleic acid may be purified from at least 2, 5,10, 15, 20, 25, 50, 100, 200 or 1000 samples. In still a furtherembodiment, a method for the invention is automated. For instance, one,two or more steps of a method according to the invention can beperformed by a robot or a microfluidic device.

In a further embodiment a method is provided for purifying nucleic acidmolecules from whole blood or from blood cells, comprising (a)contacting sample comprising whole blood or blood cells with adenaturing solvent comprising phenol; (b) adding a binding agent to thesample, wherein the binding agent comprises a chaotropic salt, analcohol or a combination thereof; and (c) contacting the sample with asilica substrate thereby binding the nucleic acid to the silicasubstrate, wherein the sample comprises an aqueous and an organic phaseafter the addition of the denaturing solvent and wherein the aqueous andorganic phases are not separated prior to binding the nucleic acid tothe silica substrate. In some aspects, a method further comprisessedimenting (e.g., by centrifugation) the sample and denaturing solventto remove excess cell debris.

In still a further embodiment an automated method is provided forpurifying nucleic acid molecules from a sample, comprising (a)contacting a nucleic acid sample with a denaturing solvent comprisingphenol; (b) adding a binding agent to the sample, wherein the bindingagent comprises a chaotropic salt, an alcohol or a combination thereof;and (c) contacting the sample with a silica bead (e.g., a magnetic bead)thereby binding the nucleic acid to the bead, wherein the samplecomprises an aqueous and an organic phase after the addition of thedenaturing solvent and wherein the aqueous and organic phases are notseparated prior to binding the nucleic acid to the bead. In furtheraspects, steps (a), (b) and/or (c) are performed by a robot. In arelated aspects, a computer-readable medium is provided comprising aprogram for instructing a robot to perform steps (a), (b) and/or (c) ofa method of the embodiments.

In yet a further embodiment a method is provided for selectivelypurifying small RNA molecules, comprising (a) contacting a nucleic acidcontaining sample with a denaturing solvent comprising phenol; (b)adding a binding agent to the sample, wherein the binding agentcomprises a chaotropic salt, an alcohol or a combination thereof; (c)contacting the sample with a silica substrate thereby binding thenucleic acid to the silica substrate; and (d) selectively eluting smallRNA molecules from the silica substrate with an elution buffer (e.g., abuffer comprising at least one chaotropic agent and at least onealcohol). In some aspects, the method further comprises (e) binding theeluted small RNA molecules to a second silica substrate in the presenceof a further binding buffer; and, optionally, (f) eluting the small RNAmolecules from the second silica substrate. Thus, in some aspects, amethods of the embodiments comprise selectively purifying or isolatingsmall RNA molecules such as miRNAs, siRNAs, piRNAs and/or syntheticRNAs.

In still a further embodiment, the invention provides kits forpurification of nucleic acid. In one aspect, a kit may comprise: adenaturing solvent comprising phenol; at least a first binding agent;and a silica substrate (e.g., magnetic beads). In certain aspects, a kitcomprises instructions for binding nucleic acid to a silica substratewithout one or more of the following steps prior to the binding: (i)precipitating nucleic acid; (ii) separating the organic and aqueousphases of a suspension or (iii) centrifugation of the sample orsuspension. In still further embodiments, a kit according to theinvention comprises a substantially aqueous binding agent and/or asubstantially alcohol binding agent. Additional compositions that may becomprised in a kit of the invention include, but are not limited to,columns; one or more further organic solvents; one or more wash agent;one or more elution buffer; a phase separating agent (e.g., chloroform);DNase enzymes, one or more detergent, one or more column (e.g., a set ofcolumns arrayed in a 96-well format), a proteinase; a reference sampleand/or storage tubes. In certain aspects, components of a kit of theinvention may be nuclease-free.

In a further embodiment, there is provided a reagent comprising (i) adenaturing solvent (e.g., a solvent comprising phenol); (ii) a bindingagent (e.g., comprising a chaotropic salt, an alcohol or a mixturethereof); and (iii) a silica substrate. For example, a reagent of theembodiments can comprise a mixture of a denaturing solvent and a bindingagent and a suspension of silica beads. Such a regent can, in someaspects, be packaged in a bottle, a vial or column. Kits comprising areagent of the embodiments are also provided.

As used herein, “a” or “an” may mean one or more. As used herein in theclaim(s), when used in conjunction with the word “comprising”, the words“a” or “an” may mean one or more than one.

The use of the term “or” in the claims means “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” As used herein “another” may mean atleast a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1: A non-limiting exemplary protocol for isolation of nucleic acidaccording to the invention.

FIG. 2: Visualization of RNA isolated in the studies of Example 1following electrophoresis on a 1% agarose gel. Treatment conditions forthe samples loaded in lanes 1-11 (from left to right) are as follows (1)1 KB ladder; (2) RNAzol®+H₂O+isopropanol; (3) RNAzol®+isopropanol; (4)RNAzol®+H₂O+ethanol; (5) RNAzol®+ethanol; (6) RNAzol®-control; (7)TRIreagent®+isopropanol; (8) TRIreagent®+chloroform+ethanol; (9)TRIreagent®+ethanol; (10) TRIreagent®-control; (11) Quick-RNA™-control,see also Table 1.

FIG. 3: Visualization of nucleic acid isolated in the studies of Example2 following electrophoresis on a 1% agarose gel. Treatment conditionsfor the samples loaded in lanes 1-11 (from left to right) are as follows(1) 1 KB ladder; (2) buffer D; (3) buffer A; (4) buffer B; (5) columnflow-through from lane 4 sample; (6) buffer C; (7) column flow-throughfrom lane 6 sample; (8) ethanol; (9) isopropanol; (10) 1 KB ladder; (11)buffer C+ethanol, see also Table 2.

FIG. 4A-B: Reproducible automated sample processing. Isolation of RNAfrom phenol-containing sample lysates, without phase separation, allowsfor direct binding of nucleic acids to a column matrix or bead and forautomated processing. A, Direct-zol™ MagBead RNA Isolation. Graph showsa comparison between manual and automated (Freedom EVO®, Tecan) sampleprocessing by nucleic acid binding directly from a phenol-containingdenaturing reagent using the Direct-zol™-96 MagBead RNA across a 96-wellplate. In each case, RNA was purified from human epithelial cells(5×10⁵/well). B, RNA was purified from human epithelial cells using theDirect-zol™-96 MagBead RNA on a Freedom EVO® automated system. RNAquality was assessed using a Bioanalyzer. RNA integrity number (RIN)demonstrates the high quality of the isolated RNA.

FIG. 5A-B: Isolation of small RNAs. Direct binding of RNA and small RNAsfrom phenol-containing lysates without phase separation, increasesrecovery of small RNAs compared to the conventional method (i.e., withphase separation/precipitation). A, Graph shows direct RNA isolationfrom phenolic extracts using a column capture, without phase separation(gray bars) versus a conventional phase separation method (lights bars).Methods of the embodiments show ˜4× higher recovery of RNAs<40 nt ascompared to a conventional phase separation method. B, Efficient smallRNA isolation using a bead capture. Small RNA was recovered with theDirect-zol™-96 MagBead RNA system and gel-separated and imaged using aBioanalyzer. Sample 1, indicates small RNA recovery from total RNA.Sample 2, indicates small RNA alone. Both samples were spiked withsmall-RNA ladder prior to Direct-zol™ MagBead purification.

FIG. 6: Isolation of nucleic acid from blood samples. Both DNA and RNAcan be efficiently isolated from phenol-containing whole-blood lysatesdirectly, without phase separation. Gel shows nucleic acids efficientlyisolated from whole blood in TRI Reagent without a phase separation. 100μl whole blood (pig) was lysed in 300 μl TRI Reagent, cleared bycentrifugation and nucleic acids bound to silica beads (Direct-zol™MagBead system).

FIG. 7: Isolation of RNA from human cells is shown. High quality RNA isisolated from different binding buffers comprising different alcohols ormixtures of alcohols or Dioxane without phase separation.

FIG. 8: Isolation of RNA from human cells is shown. High quality RNA isisolated from different binding buffers comprising different chaotropicsalts.

DETAILED DESCRIPTION

The instant invention provides an efficient method for nucleic acidisolation from samples. In particular, methods of the invention allowthe binding of nucleic acids directly from an organic sample suspensionto a silica substrate. For example, by adding an aqueous binding bufferto an organic suspension DNA can be efficiently bound to a silica matrixand purified away from contaminating protein and cell debris. Likewise,an alcohol based binding buffer can be used to directly bind RNA (andDNA) to silica from an organic suspension. Thus, the methods of theinstant invention can be used to purify RNA (including small RNAmolecules such as siRNA and miRNA), DNA, DNA and RNA or topreferentially purify RNA and/or DNA

The direct binding of nucleic acids from an organic suspension offers anumber of advantages relative to other protocols for purification.First, the organic and aqueous phases of a suspension do not need to beseparated, which is time consuming and typically involves centrifugationof the suspension and laborious removal of the aqueous (or organic)phase. Moreover, because any residual phenol contamination can inhibitthe effectiveness of downstream sample treatments (e.g., nucleic acidsequencing), one or more additional step involving extracting the samplewith a further organic compound is often required to reduce phenolcontamination. Second, there is no need to precipitate nucleic acid outof solution. Again, this process is labor intensive and, in the case ofsamples containing small amounts of nucleic acid, can result in almostcomplete loss of the sample's nucleic acid. The direct binding ofnucleic acids also has the advantage of reducing the opportunity fornuclease attack of the nucleic acids from the sample. In particular,because the methods of the invention do not involve additionalprecipitation and centrifugation steps in aqueous buffers the periodover which the nucleic acids could be exposed to any activecontaminating nuclease is reduced. Likewise, because the sample does notneed to be transferred to multiple containers the chance of importingexogenous nuclease is reduced. The foregoing advantages of the newpurification methods make them ideal for modern high throughputprotocols which require reduced labor input.

I. GENERAL PROTOCOL

An illustrative and non-limiting protocol for nucleic acid purificationaccording to the invention is exemplified below.

A. Sample Extraction

Samples may be processed for example, by freeze-thaw, proteinasetreatment or mechanical homogenization prior to organic extraction.Organic extraction may be accomplished with a protein denaturing reagentsuch as a phenol composition or acidic phenol (carbolic acid)composition with guanidinium thiocyanate, to form a suspension. Proteindenaturing reagents may additionally comprise components such as a phaseseparating agent (e.g., chloroform), an antioxidant (e.g.,2-mercaptoethanol or lipoic acid) or a chelating agent. An examplecomposition can comprise phenol:guanidinium thiocyanate:othercomponent(s) (at a ratio of 5:3:2). Once the denaturing solvent isadded, the sample may be further homogenized, for example, by vigorousshaking or blending to solubilize all possible cell components. Anorganic and aqueous phase may form in a sample which comprises phenol,but such phase separation is not required for purification.

B. Addition of Binding Agent

A binding agent is added to the sample. In particular the binding agentcomprises a chaotropic salt, an alcohol, Dioxane or a mixture thereofthat facilitates nucleic acid binding to a silica substrate.

In the case of DNA, the binding agent is a substantially aqueous agentwith chaotropic salt(s). A binding agent may also comprise additionalbuffer agents, salts, detergents and/or alcohol (e.g., lower alcoholssuch as ethanol or isopropanol). For example, the binding agent maycomprise 4-5 M guanidinium thiocyanate (GTC), 5-20% isopropanol, 2-10%glycerol and detergents. In another example, the binding agent comprises4-5 M GTC, 0.1-1.0 M sodium acetate and, optionally, detergents. One tosix volumes of the binding agent are typically added to one volume of aphenol containing sample (e.g., 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6 volumesof binding agent:sample+phenol).

In the case of RNA (or RNA/DNA), the binding agent is a substantiallyalcohol agent, such as ethanol or isopropanol. After addition of asubstantially alcohol agent the total concentration of alcohol in thesample is typically raised to greater than about 20%. For embodimentswhere binding of both DNA and RNA is desired a binding buffer maycomprise both a chaotropic salt, such as GTC, and an alcohol.

C. Nucleic Acid Binding to Silica

The sample comprising the binding agent is then contacted to a silicasubstrate. If an aqueous phase has formed in the sample then the aqueousphase alone may be contacted with the silica substrate. However,separation of organic and aqueous phases is not required for binding.The term “silica” as used herein refers materials comprising a build-upof silicon and oxygen. Such materials include, without limitation,silica, silicon dioxide, silica gel, fumed silica gel, diatomaceousearth, celite, talc, quartz, glass, glass particles including alldifferent shapes of these materials. Particles, for example, maycomprise particles of crystalline silica, soda-lime glasses,borosilicate glasses, and fibrous, non-woven glass. In certain aspects,a silica bead may be a magnetic silica bead (see, e.g., PCT PatentPubln. WO 98/31840, incorporated herein by reference).

Following binding of the nucleic acid the remaining sample suspension isremoved by, for example, pipette, vacuum or centrifugation.

D. Optional Addition of a Second Binding Agent

In certain cases, DNA can be bound to silica, while the majority of RNAremains unbound in the sample. In this case, the remaining sample can becollected and a second binding agent added. The binding agent can be asubstantially alcohol agent or Dioxane agent for binding RNA such that,after addition, the total concentration of alcohol in the sample israised to greater than about 20%, such as to a concentration of about50% or higher.

E. Optional Second Binding to Silica

The sample and binding agent are contacted with a second silicasubstrate (as above) to bind additional remaining nucleic acid, such asRNA, from the sample. The remaining sample suspension is then removed asin step 3. The remaining sample may be held for further analysis, suchas protein or lipid analysis, or discarded.

F. Wash of Silica Substrate with Bound Nucleic Acid

The silica substrate (and/or second silica substrate) comprising boundnucleic acid is washed one or more times with a washing agent. Washingagents may comprise, for example, solutions comprising alcohol, salts,buffering agents and/or detergents, that do not elute substantialamounts of nucleic acid from the silica. For example, a wash buffer maycomprise about or greater than about 40%, 50%, 60%, 70%, 80% or 90%alcohol. In certain aspects the alcohol is a volatile alcohol, such asethanol. Example washing agents include, but are not limited to, asolution of 20%-50% ethanol and 20%-50% isopropanol; a solution of about0.1-4.0 M guanidinium hydrochloride, detergents and up to about 80%ethanol; or a solution of about 80% ethanol.

G. Elute Nucleic Acid from the Silica Substrate

The silica substrate (and/or second silica substrate) comprising boundnucleic acids is contacted with an elution buffer to remove the boundnucleic acid into solution. Elution buffers typically comprise a pHbuffer agent, limited levels of salts and/or chelating agents. Buffermay additionally comprise nuclease inhibitors.

Following elution nucleic acids may either be further purified or useddirectly in down-stream analysis such as hybridization or sequencing.

II. REAGENTS AND KITS

Kits may comprise suitably aliquoted reagents of the present invention,such as an acid-phenol denaturing solvent, one or more binding agent anda silica substrate. Additional components that may be included in a kitaccording to the invention include, but are not limited to, one or morewash buffer including a magnetic bead (i.e., magnetic silica beads suchas MagBinding Beads) Pre-wash buffer, an elution buffer, a proteinasecomposition, DNase and/or RNase inhibitors, DNase or RNase enzymes,oligonucleotide primers, reference samples (e.g., samples comprisingknown amounts of DNA or RNA), distilled water, DEPC-treated water,probes, sample vials, polymerase, magnetic binding beads (e.g., magneticsilica beads such as MagBinding Beads), 96-well silica plates, 96-wellcollection plates, cover foils for 96 well plates and instructions fornucleic acid purification. In certain further aspects, additionalreagents for DNA and/or RNA clean-up may be included.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing reagent containers in close confinement for commercialsale. Such containers may include cardboard containers or injection orblow-molded plastic containers into which the desired vials areretained.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being preferred. However, the components of the kit maybe provided as dried powder(s). When reagents and/or components areprovided as a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. It is envisioned that the solvent mayalso be provided in another container means.

III. EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 RNA Isolation from Human Cells

Various techniques were used for purification of RNA from human cellsthat were frozen at −80° C. RNA was isolated from ˜100,000 frozen humancells resuspended in 50 μl of water. Samples in lanes 2-6 were lysed in125 μl of RNAzol® and further were added one or more of the following,water, ethanol, isopropanol (indicated as “Treatment” in the Table 1).Samples in lanes 7-10 were lysed in 200 μl TRIreagent® and further wereadded one or more of the following, chloroform, ethanol, isopropanol(indicated as “Treatment” in the Table 1.). Samples in lanes 2-5 and 7-9were processed by column purification using a silica matrix. Eachsuspension mixture including all components as indicated in “Treatment”(Table 1) was loaded onto the Zymo-Spin™ IC column (Zymo Research,Irvine Calif.), centrifuged at 12,000×g, and flow-through discarded. Thecolumn was washed once with 700 μl RNA Wash Buffer then centrifuged inan empty collection tube to remove residual ethanol. RNA was eluted fromthe column with 15 μl water. Samples 6 and 10 are controls and wereprocessed according to manufacturer's instructions, i.e., to separateaqueous and organic phase, samples were centrifuged. The aqueous phasewas then precipitated with isopropanol, RNA pellet washed twice with 70%ethanol, then resuspended in 15 μl water. Sample in lane 11 was lysed in150 μl of ZR RNA Buffer and processed according to the protocol forQuick-RNA™ MicroPrep (Catalog number R1050, Zymo Research, IrvineCalif.).

Any RNA eluted after the indicated treatments was resolved byelectrophoresis on a 1% agarose gel. Results from this study are shownin FIG. 2. Specifically, the results demonstrate that by using a bindingagent comprising alcohol (such as ethanol or isopropanol) RNA could beefficiently bound to (and later eluted from) a silica matrix directlyfrom the RNAzol® or TRIreagent® suspension (i.e., without the need fornucleic acid precipitation).

TABLE 1 Sample treatments for RNA shown in FIG. 2 Lane Label Treatment 11kb 1kb DNA marker 2 RNAzol ® + H₂O + +water (70 μl) + isopropanol (245μl) isopropanol 3 RNAzol ® + +isopropanol (175 μl) isopropanol 4RNAzol ® + H₂O + +water (70 μl) + 95% ethanol (245 μl) ethanol 5RNAzol ® + ethanol +95% ethanol (175 μl) 6 RNAzol ®/control +water (70μl) 7 TRIreagent ® + +isopropanol (250 μl) isopropanol 8 TRIreagent ® ++chloroform (50 μl) + 95% ethanol chloroform + ethanol (300 μl) 9TRIreagent ® + ethanol +95% ethanol (250 μl) 10 TRIreagent ®/control+chloroform (50 μl) + isopropanol (100 μl) 11 Quick-RNA ™- control

Example 2 DNA and RNA Isolation from Human Cells

Various techniques were used for purification of nucleic acid moleculesfrom human cells that were frozen at −80° C. Frozen cell samples from˜50,000 human cells were resuspended in 25 μl of water. All samples(lanes 2-9 and 11) were lysed in 100 μl of TriReagent. Following thelysis, a binding agent was added to the sample as indicated under“Treatment” (Table 2). The mixtures including all components asindicated in “Treatment” (Table 2) were loaded onto the Zymo-Spin™ ICcolumn (Zymo Research, Irvine Calif.), centrifuged at 12,000×g, andflow-through discarded (sample/lane 2-3, 5, 7-9, 11) or saved forfurther processing (sample/lane 4 and 6). 95% ethanol was added to theflow-through from samples/lanes 4 and 6 and the mixture was loaded ontothe a new Zymo-Spin™ IC column, centrifuged at 12,000×g, andflow-through discarded. After binding, all columns were washed once with700 μl RNA Wash Buffer then centrifuged in an empty collection tube toremove residual ethanol. RNA, DNA or DNA and RNA was eluted from thecolumn with 15 μl water. Buffer used in the experiments are as follows:Buffer A (A; 4-5 M GTC, 5-20% isopropanol, 2-10% glycerol anddetergents); Buffer B (B; 4-5 M GTC and 0.1 to 1.0 M sodium acetate);Buffer C (C; 4-5 M GTC, 0.1 to 1.0 M sodium acetate and detergents); andBuffer D (D; 1-5M GTC, 1-7M NaI, 20-60% ethanol and detergent).

Any nucleic acid that was eluted from the column was resolved byelectrophoresis on a 1% agarose gel. Results from this study are shownin FIG. 3 and demonstrate that both DNA and RNA could be directly boundto a silica matrix directly from the organic denaturing agent (such asTRIreagent®) suspension, without the need for nucleic acidprecipitation. Furthermore, the results demonstrate that by usingbinding agents having different compositions DNA and RNA can besequentially purified from sample. See, for example lanes 4 and 5 or 6and 7 which show DNA and RNA isolated from the same sample,respectively.

TABLE 2 Sample treatments for results shown in FIG. 3 Lane LabelTreatment 1 1 kb 1 kb DNA marker 2 D +Buffer D (375 μl) 3 A +Buffer A(375 μl) 4 B +Buffer B (375 μl) 5 B/FT Sample/lane 4 columnflow-through + 95% ethanol (500 μl) 6 C +Buffer C (375 μl) 7 C/FTSample/lane 6 column flow-through + 95% ethanol (500 μl) 8 Ethanol +95%ethanol (125 μl) 9 Isopropanol +Isopropanol (125 μl) 10 1 kb 1 kb DNAmarker 11 B + Ethanol +Buffer B (250 μl) + 95% ethanol (375 μl)

Example 3 Automated Nucleic Acid Isolation using Magnetic Beads

Isolation of RNA (DNA) from phenol-containing sample lysates, withoutphase separation, allows for direct binding of nucleic acids to a columnmatrix or beads, such as magnetic beads, and is ideal for automatedsample processing. As an example of such automated processing sampleswere processed as detailed below the resulting RNA quantified and assessfor quality.

1. 5×10⁵ human epithelial cells were lysed in TRI Reagent and cleared bycentrifugation.

2. Direct-zol™ Binding Buffer (see above) were added to the samplelysate (1:1 ratio) and mixed.

3. Magnetic silica beads (MagBinding Beads) were then added to themixture to bind nucleic acids.

4. Beads are washed by 95-100% ethanol, Direct-zol™ MagBinding BeadPreWash, and 95-100% ethanol again, several times.

5. Optionally, during the wash steps, DNase I treatment can be performedwith provided DNase I/10× Reaction Buffer.

6. Beads are dried and DNase/RNase-Free Water is added to elute RNA orDNA/RNA.

As shown in FIG. 4 methods of the embodiments proved highly effective inthe context of automated sample processing. FIG. 4A demonstrates thatreproducibly high concentration, high yield RNA can be obtained byautomated sample processing according to the embodiments. Likewise, asshown in FIG. 4B the RNA obtained is of reproducibly high quality andshows little or no significant degradation.

Example 4 Small RNA Molecule Recovery

Experiments were undertaken to assess the efficiency of isolating smallRNAs using methods of the embodiments. An example protocol for small RNAisolation is shown below:

1. RNA from human epithelial cells or mouse liver tissue homogenized inTRI Reagent was isolated by Direct-zol™ method and compared to theconventional phase separation. RNA was analyzed and quantified by aSmall RNA Chip (Bionalyzer; FIG. 5).

2. A mixture of small RNA oligonucleotides (ZR small-RNA ladder) wasrecovered directly or “spiked” into a total RNA and recovered by theDirect-zol™ MagBinding Bead protocol. RNA was analyzed and quantified bya Small RNA Chip (Bioanalyzer; FIG. 5).

Results of the studies shown in FIG. 5A demonstrate that direct RNAisolation from phenolic extracts, without phase separation, shows higherrecovery of RNAs<40 nt (4 fold higher) as compared to a conventionalphase separation method. Again recovered RNA was shown to be of highquality (FIG. 5B).

Example 5 Nucleic Acid Extraction from Whole Blood

Both DNA and RNA can be isolated from phenol-containing whole-bloodlysates directly, without phase separation. For these studies 100 ul ofwhole-blood (pig or human) was lysed in 300 ul TRI Reagent BD (TRIReagent BD is for blood, plasma, serum) and cleared by centrifugation.The lysate/supernatant is aspirated and dispensed into a new tube. Thelysate/supernatant can be processed either by spin-column/spin-plate(Direct-zol™ RNA MiniPrep, Direct-zol™-96 RNA) or magnetic silica beadformat (Direct-zol™ MagBinding Bead RNA) as detailed below:

1. For spin-column/spin-plate: 95% ethanol is added to thelysate/supernatant (1:1 ratio) and the mixture is loaded into thecolumn. Column is then washed by Direct-zol™ RNA PreWash and RNA WashBuffer several times and RNA is eluted with DNase/RNase-Free Water.

2. For the silica magnetic bead format: Direct-zol™ Binding Buffer isadded to the lysate/supernatant (1:1 ratio) and mixed. Magnetic silicabeads (MagBinding Beads) are then added to the mixture to bind nucleicacids. Beads are washed by 95-100% ethanol, Direct-zol™ MagBead PreWash,and 95-100% ethanol again, several times. During the wash steps, DNase Itreatment can be performed with provided DNase I/10× Reaction Buffer.Beads are dried and DNase/RNase-Free Water is added to elute the RNA orDNA/RNA.

Results of the whole blood extraction are shown in FIG. 6 anddemonstrate highly efficient isolation of RNA and DNA from the samples.

Example 6 Selective Isolation of Small RNAs

Studies were undertaken to determine if small RNA molecules could beselectively enriched using the methods of the embodiments. An exampleprotocol for such selective enrichment is shown below:

1. Human epithelial cells were lysed in TRI Reagent and cleared bycentrifugation.

2. 95% ethanol is then added to the sample lysate (1:1 ratio) and themixture is loaded into the column.

3. small-RNA Elution Buffer, which comprises at least one chaotropicsalt and at least one alcohol was added to the column. Thisremoves/elutes small RNAs from the column (<200 nt).

4. To the flow-through that contains the small-RNAs (<200 nt), add 1volume 95% ethanol and load the mixture into a new column.

5. Column is then washed by Direct-zol™ RNA PreWash and RNA Wash Buffer(see above) several times and RNA is eluted with DNase/RNase-Free Water.

Results of these studies demonstrate that small RNAs could beselectively purified relative to larger mRNA and rRNA components.

Example 7 Isolation of RNA from Human Cells from Tri-Reagent withDifferent Binding Buffers

RNA was isolated from about 50,000 human cells resuspended in 25 μlwater. All samples were lysed in 100 ml TRI Reagent and further agentswere added as follows:

(1) One volume (125 μl) of either 100% methanol, ethanol, isopropanol,n-butanol, 3-methyl-1-butanol or a mixture thereof (FIG. 7) or (2) onevolume (125 μl) of a chaotropic salt or a buffer containing chaotropicsalt, followed by two volumes (i.e. 250 μl) of 95% ethanol (FIG. 8).

Each mixture was loaded onto a separate spin Column (Zymo-Spin—IC, ZymoResearch Corp.), centrifuged at 12,000×g with flow through beingdiscarded after. Each column was then processed according to theprotocol for the Direct-zol™ RNA miniprep, from step 3 on (Cat. No.82050, Zymo Research Corp.). RNA was eluted with 15 μl DNAse/RNAse-freewater. High-quality RNA is evident from all alcohols or Dioxane orchaotropic salts tested (FIG. 7 and FIG. 8, agarose gel electrophoresisand spectrophotometric analysis is shown below In Tables 3 and 4).

TABLE 3 Results for RNA binding with a variety of alcohol-based buffersBinding buffer component ng/μl 260/280 260/230 methanol 104.57 2.01 2.14ethanol 130.51 2.02 2.19 isopropanol 132.02 2.02 2.26 n-butanol 75.482.04 2.21 3-methyl-1-butanol (IAA) 53.13 1.98 2.00 methanol/ethanol50/50 108.99 2.01 2.12 isopropanol/ethanol 50/50 133.24 2.03 2.26n-butanol/ethanol 50/50 136.75 2.03 2.23 IAAl/ethanol 50/50 145.40 2.022.25 1,4-Dioxane 126.07 2.02 2.2

TABLE 4 Results for RNA binding with a variety of chaotropic saltsBinding buffer component ng/μl 260/280 260/230 Binding buffer with GTC107.32 2.07 2.18 Binding buffer with NaI 112.04 2.05 1.28 GHCl (6M)130.82 2.06 2.2 Urea (9M) 105.22 2.02 2.05 Thiourea (1.8M)) 106.44 2.051.81 NaI (7M) 87.17 2.02 2

REFERENCES

Each of the foregoing documents is hereby incorporated by reference inits entirety:

U.S. Pat. Nos. 4,843,155; 5,472,872; 6,210,945

U.S. Patent Publication 20100222560

PCT Patent Publn. WO 98/31840.

Sambrook et al., In: Molecular Cloning—A Laboratory Manual, 2001.

1. A method for binding a nucleic acid to a silica substrate forpurification comprising: (a) contacting sample comprising whole blood orblood cells with a denaturing solvent comprising phenol; (b) adding abinding agent to the sample, wherein the binding agent comprises achaotropic salt, a lower alcohol or a mixture thereof; and (c)contacting the sample with a silica substrate thereby binding thenucleic acid to the silica substrate, wherein the nucleic acid in thesample is substantially unprecipitated prior to binding of the nucleicacid to the silica substrate, and wherein the nucleic acid is bound tothe silica substrate with out substantially separating the organic andaqueous phases in the sample.
 2. The method of claim 1, wherein thedenaturing solvent comprises at least one additional agent selected fromthe group consisting of a chaotropic salt, an antioxidant, a chelatingagent, a phase separating agent and a buffer. 3-4. (canceled)
 5. Themethod of claim 1, wherein the denaturing solvent has a pH of betweenabout 6.0, 5.5, 5.0, 4.5 or 4.0 and about 2.0.
 6. (canceled)
 7. Themethod of claim 1, wherein the method is automated.
 8. The method ofclaim 1, wherein the binding agent is added to the sample before thedenaturing solvent.
 9. The method of claim 1, wherein the silicasubstrate is added to the sample before the binding agent.
 10. Themethod of claim 1, wherein steps (a) and (b) are performedcontemporaneously.
 11. The method of claim 10, wherein steps (a), (b)and (c) are performed contemporaneously. 12-14. (canceled)
 15. Themethod of claim 14, wherein the binding agent comprises a lower alcoholselected from the group consisting of methanol, ethanol, isopropanol,butanol, and a combination thereof.
 16. The method of claim 12, whereinthe total alcohol content of the sample is at least about 40% after thebinding agent is added.
 17. The method of claim 1, wherein the bindingagent is an aqueous solution comprising a chaotropic salt or mixture ofchaotropic salts.
 18. The method of claim 17, wherein the chaotropicsalt is guanidinium thiocyanate, guanidinium chloride, sodium iodide,sodium perchlorate, urea or thiourea.
 19. The method of claim 17,wherein the binding agent comprises pH buffer.
 20. The method of claim1, wherein steps (b) and (c) are performed contemporaneously.
 21. Themethod of claim 1, wherein the nucleic acid is DNA.
 22. The method ofclaim 1, wherein the nucleic acid is RNA.
 23. The method of claim 22,wherein the nucleic acid is miRNA or siRNA.
 24. The method of claim 1,wherein the silica substrate is in the form of beads, fibers or a porousmatrix.
 25. The method of claim 1, wherein the silica substrate is a(para)magnetic bead. 26-27. (canceled)
 28. The method of claim 1,further comprising washing the silica substrate with a wash solutionafter binding the nucleic acid to the silica substrate.
 29. The methodof claim 28, wherein the wash solution comprises a salt, a chaotropicsalt, a detergent or an alcohol.
 30. The method of claim 1, furthercomprising eluting the nucleic acid from the silica substrate with anelution buffer after binding the nucleic acid to the silica substrate.31-90. (canceled)
 91. A kit for nucleic acid purification comprising: adenaturing solvent comprising phenol; a binding agent comprising achaotropic salt, an alcohol or a combination thereof; and a silicasubstrate. 92-94. (canceled)